Neonatal Compartment Syndrome Secondary to a Failed Peripheral Inserted Central Catheter Insertion: A Case Report.

Introduction: Neonatal compartment syndrome is a rare phenomenon with a limited number of cases reported in the literature with varying etiologies. Current literature categorizes etiologies as either intrinsic or extrinsic. To the best of our knowledge, difficult delivery and delivery through vacuum are the only two iatrogenic etiologies that have been reported in the literature. Thus, this may be the first reported case of neonatal compartment syndrome secondary to a failed peripherally inserted central catheter (PICC) insertion. Case Report: We present a case of a pre-mature neonate with diffuse discoloration, paralysis, and loss of palpable pulses of the right upper extremity after a failed PICC insertion. The clinical features led to a diagnosis of compartment syndrome. Interventions were not carried out due to the pre-maturity and instability of the patient. The patient passed away at 38 days of age due to refractory hypotension and patent ductus arteriosus. Conclusion: We present a case of neonatal compartment syndrome caused by a previously unreported etiology, highlighting the current dearth of knowledge. Clinicians should be aware of the unique clinical presentation of neonatal compartment syndrome and maintain high suspicion even without an obvious etiology.

Operative Treatment of a Patient with a Pathological Fracture through a Large Non-ossifying Fibroma: A Case Report.

Introduction: Non-ossifying fibroma (NOF) is a common benign fibrogenic bone lesion commonly found in the metaphysis of long bones. While small NOFs are typically asymptomatic and left untreated, large NOFs are often associated with pathologic fractures that can be treated conservatively or operatively. To our knowledge, the NOF presented in this case report is one of the largest reported in the literature. Case Report: We present a case of a 12-year-old Hispanic female who presented to our institution after falling off a horse and landing on her right leg and wrist. Radiographs revealed a right distal femur fracture through an unusually large lytic bone lesion. The patient was treated with curettage, grafting, open reduction, and internal fixation, given the unstable and pathological nature of the fracture. Conclusion: This case substantiates the literature that large fractures through a NOF can be appropriately managed operatively. However, orthopedic surgeons should be aware of the risks of surgery, including infection, when considering operative management of patients who present with large unstable pathologic fractures through a NOF.

An injury-responsive mmp14b enhancer is required for heart regeneration.

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.

ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment.

Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment.

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mislocalized, prompting us to investigate the scope of the role of Mesp1 in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.

An enhancer-based gene-therapy strategy for spatiotemporal control of cargoes during tissue repair.

The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.

Calcium pyrophosphate dihydrate crystals in a 9-year-old with osteomyelitis of the knee: A case report.

BACKGROUND: Calcium pyrophosphate dihydrate deposition disease (CPPD), or pseudogout, is an inflammatory arthritis common among elderly patients, but rarely seen in patients under the age of 40. In the rare cases presented of young patients with CPPD, genetic predisposition or related metabolic conditions were almost always identified. CASE SUMMARY: The authors report the case of a 9-year-old boy with no past medical history who presented with acute knee pain and swelling after a cat scratch injury 5 d prior. Synovial fluid analysis identified calcium pyrophosphate dihydrate crystals. Further MRI analysis identified osteomyelitis and a small soft tissue abscess. CONCLUSION: This case presents the extremely rare diagnostic finding of calcium pyrophosphate dihydrate crystals in a previously healthy pediatric patient. The presence of osteomyelitis presents a unique insight into the pathogenesis of these crystals in pediatric patients. More research needs to be done on the role of CPPD in pediatric arthritis and joint infection.

Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor.

The low-density lipoprotein receptor (LDLR) controls cellular delivery of cholesterol and clears LDL from the bloodstream, protecting against atherosclerotic heart disease, the leading cause of death in the United States. We therefore sought to identify regulators of the LDLR beyond the targets of current therapies and known causes of familial hypercholesterolemia. We found that cold shock domain-containing protein E1 (CSDE1) enhanced hepatic LDLR messenger RNA (mRNA) decay via its 3' untranslated region and regulated atherogenic lipoproteins in vivo. Using parallel phenotypic genome-wide CRISPR interference screens in a tissue culture model, we identified 40 specific regulators of the LDLR that were not previously identified by observational human genetic studies. Among these, we demonstrated that, in HepG2 cells, CSDE1 regulated the LDLR at least as strongly as statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. In addition, we showed that hepatic gene silencing of Csde1 treated diet-induced dyslipidemia in mice to a similar degree as Pcsk9 silencing. These results suggest the therapeutic potential of targeting CSDE1 to manipulate the posttranscriptional regulation of the LDLR mRNA for the prevention of cardiovascular disease. Our approach of modeling a clinically relevant phenotype in a forward genetic screen, followed by mechanistic pharmacologic dissection and in vivo validation, may serve as a generalizable template for the identification of therapeutic targets in other human disease states.

Transcription Factor GATA4 Regulates Cell Type-Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitors.

BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

Transcriptional regulation of vascular smooth muscle cell proliferation, differentiation and senescence: Novel targets for therapy.

Vascular smooth muscle cells (SMC) possess a unique cytoplasticity, regulated by transcriptional, translational and phenotypic transformation in response to a diverse range of extrinsic and intrinsic pathogenic factors. The mature, differentiated SMC phenotype is physiologically typified transcriptionally by expression of genes encoding "contractile" proteins, such as SMα-actin (ACTA2), SM-MHC (myosin-11) and SM22α (transgelin). When exposed to various pathological conditions (e.g., pro-atherogenic risk factors, hypertension), SMC undergo phenotypic modulation, a bioprocess enabling SMC to de-differentiate in immature stages or trans-differentiate into other cell phenotypes. As recent studies suggest, the process of SMC phenotypic transformation involves five distinct states characterized by different patterns of cell growth, differentiation, migration, matrix protein expression and declined contractility. These changes are mediated via the action of several transcriptional regulators, including myocardin and serum response factor. Conversely, other factors, including Kruppel-like factor 4 and nuclear factor-κB, can inhibit SMC differentiation and growth arrest, while factors such as yin yang-1, can promote SMC differentiation whilst inhibiting proliferation. This article reviews recent advances in our understanding of regulatory mechanisms governing SMC phenotypic modulation. We propose the concept that transcription factors mediating this switching are important biomarkers and potential pharmacological targets for therapeutic intervention in cardiovascular disease.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

The Relationship Between Body Mass Index and the Magnitude of Curve at Diagnosis of Adolescent Idiopathic Scoliosis: A Retrospective Chart Review.

Purpose: To evaluate the relationship between body mass index (BMI) and spinal curvature in patients presenting with idiopathic scoliosis at a major pediatric tertiary care centre. Patients and Methods: Retrospective chart review (2015-2019). Data extracted from patient's first visit included age, sex, height, weight, spinal curvature (magnitude, location), referral source, physical activity participation (yes/no), pain (yes/no). Demographics were analyzed descriptively. The relationship between BMI and spinal curve magnitude was analyzed using Spearman correlation coefficient. Linear regression was applied to determine the relationship between BMI, curve magnitude, and curve location. Exploratory univariate analyses were conducted for BMI and referral source, pain, and skeletal maturity, and physical activity and pain. Results: A total of 206 patient charts were included (177 females, 29 males). Patients presented with double major (41.3%), thoracic (26.7%), thoracolumbar (22.8%), and lumbar (9.2%) curves. Mean (SD) BMI percentile was 48.3 (30.5). No relationship existed between BMI percentile and curve magnitude with curve locations combined. However, a test for interaction revealed a positive relationship between BMI percentile and curve magnitude for adolescents with double major curves, and a negative relationship for adolescents with thoracic curves. Exploratory analyses suggested a relationship between BMI percentile and presence of pain, and between referral source and curve magnitude. No relationship was observed between BMI and skeletal maturity, or physical activity and pain. Conclusion: The relationship between BMI varied by curve location within this cohort, in which most patients presented with a BMI <85th percentile. Findings highlight the importance of sensitive history taking and careful physical examination for early detection of scoliosis.

Transcription factor protein interactomes reveal genetic determinants in heart disease.

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.

Comprehensive cell type decomposition of circulating cell-free DNA with CelFiE.

Circulating cell-free DNA (cfDNA) in the bloodstream originates from dying cells and is a promising noninvasive biomarker for cell death. Here, we propose an algorithm, CelFiE, to accurately estimate the relative abundances of cell types and tissues contributing to cfDNA from epigenetic cfDNA sequencing. In contrast to previous work, CelFiE accommodates low coverage data, does not require CpG site curation, and estimates contributions from multiple unknown cell types that are not available in external reference data. In simulations, CelFiE accurately estimates known and unknown cell type proportions from low coverage and noisy cfDNA mixtures, including from cell types composing less than 1% of the total mixture. When used in two clinically-relevant situations, CelFiE correctly estimates a large placenta component in pregnant women, and an elevated skeletal muscle component in amyotrophic lateral sclerosis (ALS) patients, consistent with the occurrence of muscle wasting typical in these patients. Together, these results show how CelFiE could be a useful tool for biomarker discovery and monitoring the progression of degenerative disease.

Control of ribosomal protein synthesis by the Microprocessor complex.

Ribosome biogenesis in eukaryotes requires the coordinated production and assembly of 80 ribosomal proteins and four ribosomal RNAs (rRNAs), and its rate must be synchronized with cellular growth. Here, we showed that the Microprocessor complex, which mediates the first step of microRNA processing, potentiated the transcription of ribosomal protein genes by eliminating DNA/RNA hybrids known as R-loops. Nutrient deprivation triggered the nuclear export of Drosha, a key component of the Microprocessor complex, and its subsequent degradation by the E3 ubiquitin ligase Nedd4, thereby reducing ribosomal protein production and protein synthesis. In mouse erythroid progenitors, conditional deletion of Drosha led to the reduced production of ribosomal proteins, translational inhibition of the mRNA encoding the erythroid transcription factor Gata1, and impaired erythropoiesis. This phenotype mirrored the clinical presentation of human "ribosomopathies." Thus, the Microprocessor complex plays a pivotal role in synchronizing protein synthesis capacity with cellular growth rate and is a potential drug target for anemias caused by ribosomal insufficiency.

ATAC-Seq Reveals an Isl1 Enhancer That Regulates Sinoatrial Node Development and Function.

RATIONALE: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the cis-regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified. OBJECTIVE: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function. METHODS AND RESULTS: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the Isl1 locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations. CONCLUSIONS: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) cis-regulation of Isl1 specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human ISL1 enhancer may regulate human SAN function.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

RATIONALE: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. OBJECTIVE: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. METHODS AND RESULTS: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. CONCLUSIONS: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Presynaptic Homeostasis Opposes Disease Progression in Mouse Models of ALS-Like Degeneration: Evidence for Homeostatic Neuroprotection.

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.